PROJECT SUMMARY On the morning of December 18th 2014, I arrived in the laboratory early to check on my experiment ? what ended up being the ?rst proof that the Mutagenic Chain Reaction (MCR) functioned as a highly ef?cient CRISPR/Cas-based gene-drive system in fruit ?ies [1]. I later built a similar, albeit more complex, MCR construct in mosquitoes, that was tested in collaboration with the James group (UCI) [2]. As was the case for the fruit ?y element, the mosquito MCR propagates with exceptional ef?ciency (99.5%) via the germline. During this process, my advisor Ethan Bier and I expanded the concept of `active genetics' [4] to a family of genetic elements that actively copy themselves onto the companion chromosome (as in the MCR). These elements bypass the constraints of Mendelian inheritance, thereby potentially overcoming current limitations in laboratory experiments. Gene drive systems can be used to combat vector-borne diseases thereby bene?ting global public health (e.g., malaria eradication), as well as to restore native ecosystems (e.g., suppress invasive species populations). Although I am interested in future applications in diverse ?elds, during the award period I will focus on deepening the knowledge on the mechanism of action of active genetic elements in the fruit ?y. Here I propose to build and characterize in Drosophila melanogaster three categories of active genetic elements: (1) Full MCR-gene drives, (2) Split, ?transcomplementing-MCR?, an alternative that could offer advantages when performing population modi?cation in the wild, (3) Reversal constructs to stop, limit or reverse the spread of a Cas9-based gene drive in the wild. 1) I will examine the basis for the extraordinary ef?ciency of our existing gene drive technology and re?ne its functionality for future ?eld applications. Several gene drives, based on MCR technology, will be developed in the fruit ?y. Different regulatory regions will be identi?ed to drive the expression of the Cas9 nuclease in the most effective time during development while assuring its restriction to germline cells. 2) I will build and test trans-complementing-MCRs in which the two primary MCR components (Cas9 and gRNAs) are split in two separate transgenic constructs. Each component individually would not generate inheritance bias; only when combined will these elements reconstitute a gene drive arrangement. This technology could be used in population suppression schemes where a full gene drive, purposely affecting ?tness, would otherwise render problematic the ampli?cation of the laboratory population to the levels necessary for ?eld release. 3) I will develop reversal constructs that can counteract the spread of a Cas9-based gene drive construct in a population. I will test two different types of such constructs: the ?rst one acts by cutting out and replacing the gene drive at the same locus at which it is inserted; the second type is located in a different location in the genome, but carries guide-RNAs able to exploit the Cas9 protein to disrupt the Cas9 gene itself.